ABSTRACT Streptococcus pneumoniae has remained one of the major causative agents of potentially life threatening human disease in our era. Nevertheless, introduction of antibiotics (penicillin) and [unreadable] more recently [unreadable] the conjugate vaccine provoked major evolutionary changes in the structure of natural populations of this pathogen. The purpose of this research proposal is to combine the power of whole genome sequencing (WGS) and epidemiology to obtain insights into the mechanisms by which antibiotic resistant and non-vaccine type pneumococcal lineages emerged in the in vivo environment. The studies will have three foci of concentration. Project I. Three highly penicillin resistant pneumococcal (PRPn) clones: ST-1, ST-2 and ST-3, each with a unique sequence type (MLST) and capsular type have achieved pandemic spread and appear to remain stable over long times and distant geographic sites of isolation. Such genetic stability in a highly recombinogenic pathogen is unusual and it contrasts with the well documented diversity of penicillin susceptible pneumococci. The purpose of this project is to use whole genome sequencing (WGS) to better document and understand the nature and mechanisms of genetic stability in these three PenR clones. Project II. An outbreak investigation in an AIDS hospice identified members of the PenR clone ST-1 as causative agents of the disease. All but two of the 11 single patient isolates expressed capsular polysaccharide 23F typical of this clone. However, 2 isolates produced capsular type 3 which was associated with a tremendous (106 fold) increase in virulence measured in mouse IP models. The purpose of these studies is to use WGS [unreadable] in combination with the appropriate in vitro complementation experiments [unreadable] to identify genetic determinants of virulence produced [unreadable] in vivo [unreadable] in such a spontaneous [unreadable]experiment of nature[unreadable]. Project III. Pneumococcal strains expressing the non-vaccine type (NVT) capsular polysaccharides, such as 11A, 6A or 19A have been identified as minority components of the nasopharyngeal flora of children attending Day Care Centers [unreadable] even before the introduction of the conjugate vaccine. WGS will be used to test if the emergence of these strains from minority to majority status in the nasopharynx and from colonizers to disease causing strains [unreadable] is accompanied by changes in genetic makeup